The major goals of this proposal are to determine the toxicity and potential benefits associated with the intracranial administration of two new recombinant adenovirus expressing herpes virus thymidine kinase (H5.001RSVtk) or interferon-beta (H5.010CMVINF beta) in patients with malignant gliomas. Gene therapy for brain tumors has the potential of providing significant clinical benefit beyond that achieved with currently available therapies. The major limitation to the advance of this field is the availability of safe and efficient gene therapy vectors. Adenovirus vectors are very efficient gene transfer agents but their distribution and function within tumors is not well studied in patients. We previously conducted a phase I study using a recombinant adenovirus expressing HSVtk (H5.010RSVtk, viral genome deletions in E1 and E3) in patients with advanced malignant gliomas. This study demonstrated that intracranial administration of adenovirus could be safely performed using two successive doses of vector up to 1011 PFU. The dose limiting toxicity resulted from an increase in intracranial pressure believed to be secondary to virus induced inflammation. In this initial trial we observed a durable complete response in one patient (disease free after 2 years) and prolonged freedom from progression (>10 months) in three other patients. These results are encouraging and have led to the design of two new clinical trials using a new third generation adenovirus (H5.001RSVtk, viral genome deletions in E1 and E4) and a new vector capable of delivering interferon-beta. The H5.001RSVtk vector has the advantage of being free of replication competent adenovirus which may limit its toxicity. We propose to test this vector (Aim 1) to establish that it is safer and can be administered at higher doses to enhance the efficacy of this therapy. We have developed a non-invasive positron emission tomography imaging technique to assess both the duration and distribution of thymidine kinase enzymatic activity in patients undergoing HSVtk based gene therapy (Aim 2). The H5.010CMVINF-beta vector has shown significant tumoricidal activity in animal models and will be tested in a phase I clinical study (Aim 3). This vector encodes a secreted protein that may be more effective at reaching the entire tumor. We believe that this study will have important implications for brain tumor gene therapies in general and provide a new clinical modality for assessing gene transfer.